RESEARCH ACCOMPLISHMENTS A. CHARACTIZATION OF A NOVEL INTERFERON-MEDIATED AUTOINFLAMMATORY DISEASE CAUSED BY GAIN OF FUNCITON MUTATIONS IN STING, SAVI. Using WES in an index patient, we identified that gain of function mutations in the TMEM173, the gene that encodes a key adaptor molecule in the interferon pathway, STING, causes a novel autoinflammatory disease that we named STING-associated vasculopathy with onset in infancy (SAVI). These patients have constitutive activation of the interferon-beta pathway that cannot be further up-regulated with stimulation. STING is expressed in endothelial cells, and activation of the STING pathways in these cells results in endothelial activation and apoptosis, a feature that may explain the predominant manifestation as peripheral vasculitis and vasculopathy. Several patients have developed interstitial lung disease. Constitutive up-regulation of phosphorylated STAT1 in patients' lymphocytes was reduced by JAK inhibitors. These studies indicate that constitutive STING activation can lead to vasculitis and vasculopathy and the development of interstitial lung disease and suggest that STING may be a therapeutic target for therapy in SAVI and likely other diseases that are interferon mediated. B. GAIN OF FUNCTION MUTATION IN NLRC4 CAUSE AN AUTOINFLAMMATORY SYNDROME WITH RECURRENT FEVER FLARES AND MACROPHAGE ACTIVATION (NLRC4-MAS) The identification of a de novo mutation T337S in the intracellular innate immune sensor, NLRC4 represents the first monogenic defect that is causes an intrinsic macrophage activation defect that can lead to the development of macrophage activation syndrome. The comparison with NOMID, an autoinflammatory disease caused by gain-of-function mutations in the analogous NOD-like receptor, NLRP3, is described above, and suggests a unique role of NLRC4 in causing macrophage activation syndrome. Functional analyses demonstrated spontaneous inflammasome formation and production of the inflammasome-dependent cytokines IL-1beta; and IL-18, with serum IL-18 levels exceeding levels seen in NOMID patients. The monogenic defect in the NLRC4 inflammasome drives IL-1 secretion in blood monocytes at comparable levels to those seen in NOMID. In contrast to NOMID, NLRC4-MAS patients have very high elevation of IL-18 produced in macrophages, thus providing us with a disease model to study macrophage activation. Our data suggest that IL-18 may play a critical role in the development of macrophage activation syndrome. High IL-18 levels are also seen in patients with other inflammatory conditions that are prone to the development of macrophage activation syndrome i.e Stills disease, that may share common pathways that drive macrophage activation. This project is being developed in collaboration with the Metzger Scholar, Dr. Canna. C. NOVEL INSIGHTS IN TO THE INTERFERON MEDIATED DISEASE CANDLE. Chronic atypical neutrophilic dermatosis with lipodystrophy, and elevated temperature (CANDLE) is an autoinflammatory disease caused by mutations in the proteasome subunit PSMB8. The discovery led to a unifying concept that a group of rare diseases including JMP (joint contractures, muscle atrophy and panniculitis-induced lipodystrophy) syndrome, and Nakajo Nishimura syndrome (NNS) are one disease spectrum of proteasome-assoociated autoinflammatory syndromes (PRAAS). We identified novel mutations in PSMB8 and in additional proteasome genes as the genetic causes for CANDLE. All patients expressed high IP-10 (Interferon gamma-induced protein 10), MCP-1, and other chemokines and cytokines associated with interferon induced diseases and a strong interferon (IFN) response signature (IRS) confirming that IFN dysregulation is linked to global proteasome dysfunction not related to mutations in one component. An ongoing compassionate use study with the JAK inhibitor, baricitinib (Eli Lilly) that can inhibit interferon signaling, is recruiting patients with CANDLE and SAVI and other interferonopathies who are unresponsive to IL-1 blocking agents. We are studying the inflammatory phenotype and metabolic changes in CANDLE, including hyperlipidemias, metabolic syndrome and muscle atrophy. D. UPDATES ON IL-1-MEDIATED DISEASES, NOMID and DIRA: 1. FDA submission of our long-term outcome data using the short-acting IL-1 inhibitor anakinra (SOBI) in patients with neonatal-onset multisystem inflammatory disease (NOMID) led to FDA approval of anakinra for the treatment of NOMID in December 2012. 2. We assessed the efficacy of the long acting IL-1 blocking agent canakinumab in 6 NOMID patients. At month 6, none had full remission, although 4/6 achieved normalization of inflammatory markers in the blood, but 5/6 had persistent CNS leucocytosis. At the last study visit 12-24 months into treatment, 5/6 patients achieved inflammatory remission in blood and good symptom control, but 4/6 had continued CNS leucocytosis. Visual acuity and field were stable in all patients, progressive hearing loss occurred in 1/10 ears. Adverse events (AEs) were rare. Canakinumab at the studied doses improves symptoms and serum inflammatory features of NOMID, although low-grade CNS leukocytosis in four patients and headaches in one additional patient persisted. Whether further dose intensifications are beneficial in these cases remains to be assessed. These data suggest likely differneces in drug penetration into different organs, that need to be further studied. 3. Analyses of long-term outcomes in patients with DIRA treated with the IL-1 blocking agent anakinra indicate good inflammatory control and prevention of organ damage in nine patients. E. ONGING STUDY OF PATIENTS WITH UNDIFFERENTIATED AUTOINFLAMMTORY DISEASES We evaluate and treat patients with severe inflammatory diseases of early onset in infancy. In addition to a detailed immune evaluation that includes gene expression studies, patients and their parents if indicated undergo genetic analyses including evaluation through next generation sequencing, including whole exome sequencing (WES). We attempt to identify molecular targets that allow us to better treat patients with AIDs. CONCLUSIONS AND SIGNIFICANCE 1. We identified two novel autoinflammatory diseases SAVI and NLRC4 MAS that shed light on basic disease mechanisms that cause autoinflammatory disease phenotypes. 2. The novel genetic defects in (CANDLE) and the discovery that mutations in a key adaptor molecule STING, a gatekeeper for IFN beta transcription cause SAVI, suggest a causative role of ongoing IFN signaling in leading to autoinflammatory diseases and suggest novel targets for treatment. 3. Our data provide a rational to test IFN inhibiting drugs and an ongoing compassionate use study using the JAK1/2 inhibitor baricitinib will assess the clinical efficacy and safety in patients with CANDLE and SAVI and in patients with clinical and laboratory evidence of IFN mediated disease. 4. The identification mutations in NLRC4 causing macrophage activation, lead to the exploration of the role of IL-18 in macrophage activation and suggest IL-18 as a potential target for treatment. 5. IL-1-blocking therapies are FDA-approved for the use in CAPS including NOMID based on work doe at the NIH, but differences the effectiveness in in controlling organ inflammation may exist between different IL-1 blocking agents. 6. A clinical study in patients with DIRA using the long-acting IL-1 inhibitor rilonacept (Regeneron) assesses efficacy of a long acting IL-1 inhibitor in DIRA patients. 7. We developed an integrative approach using immune evaluation, whole exome sequencing and targeted therapeutics to assess patients with undifferentiated autoinflammatory disorders, with the goal to better diagnose, understand disease pathogenesis and identify novel targets for therapy.